Cycloalkylidenol analogues of prostaglandins E and F

ABSTRACT

Analogues of prostaglandins E and F, in which the C 15  hydroxyl group is incorporated into a cycloalkylidene moiety, stimulate smooth muscle.

REFERENCE TO PRIOR APPLICATION

This is a continuation-in-part of U.S. Pat. application Ser. No.436,221, filed on Jan. 24, 1974, which in turn is a division of U.S.Pat. application Ser. No. 383,670, filed on July 30, 1973; both priorapplications are abandoned.

BACKGROUND OF THE INVENTION Field of the Invention

The compounds of this invention are derivatives or analogues of a classof naturally occurring chemical compositions known as prostaglandins.

Natural prostaglandins are twenty-carbon atom alicyclic compoundsrelated to prostanoic acid which is represented by the followingstructural formula: ##STR1## By convention, the carbon atoms of I arenumbered sequentially from the carboxylic carbon atom. An importantstereo-chemical feature of I is the trans-orientation of the side chainsC₁ -C₇ and C₁₃ -C₂₀. In I, as elsewhere in this specification, a dottedline ( ) indicates projection of the covalent bond below the plane of areference carbon atom (the alpha-configuration), while a wedged line ( )represents direction above said plane (the beta-configuration). Thesenotations are applicable to all compounds hereinafter discussed.

The twelve natural prostaglandins which have been isolated to date havethe structural formula: ##STR2## in which: L and M may be ethylene orvinylene radicals; and, the five-membered ring ##STR3## may be ##STR4##

The natural prostaglandins represented by II, are classified accordingto the functional groups present in the five-membered ring structure andthe presence of double bonds in the ring or chains. Prostaglandins ofthe F-class (PGF's) are characterized by α-oriented hydroxyl groups atC₉ and C₁₁ ; those of the E-type (PGE's) have a carbonyl group at C₉ andan α-oriented hydroxyl group at C₁₁ ; compounds of the A-series (PGA's)contain a carbonyl group at C₉ and a double bond at C₁₀ (Δ¹⁰,11 ); andmembers of the B-class (PGB's) have a carbonyl group at C₉ and anunsaturated bond between C₈ and C₁₂ (Δ⁸,12 ). Within each of the F, E,A, and B classes of prostaglandins are three subclassifications basedupon the presence of double bonds in the side chains at C₅, C₁₃, or C₁₇.The presence of a trans-unsaturated bond only at C₁₃ is indicated by thesubscript numeral 1; thus, for example, PGE₁ denotes a prostaglandin ofthe E-type (carbonyl at C₉ and an alpha-hydroxyl at C₁₁) with atrans-double bond at C₁₃. The presence of both a trans-double bond atC₁₃ and a cis-unsaturated bond at C₅ is denoted by the subscript numeral2, for example, PGE₂. Lastly, a trans-double bond at C₁₃, a cis-doublebond at C₅, and a cis-double bond at C₁₇ is indicated by the subscriptnumeral 3, for example, PGE₃. The above notations apply toprostaglandins of the A, B and F series as well, however, in the latterthe alpha-orientation of the hydroxyl group at C₉ is indicated by thesubscript Greek letter α after the numerical subscript. Thus PGF₃.sub.αrepresents 9α,11α,15α-trihydroxy-5,17-cis, 13-trans-prostatrienoic acid(utilizing nomenclature based upon prostanoic acid).

It is important to note that in all natural prostaglandins there is analpha-oriented hydroxyl group at C₁₅. In the Cahn-Ingold-Prelog systemof defining stereochemistry, this C₁₅ hydroxyl group is in theS-configuration.

11-desoxy derivatives of PGE and PGF molecules do not occur as such innature, but constitute a class of compounds which possess biologicalactivity related to the parent compounds. Formula II represents11-desoxy PGE's and PGF's when ##STR5##

I.U.P.A.C nomenclature of prostaglandins designates the carboxylic sidechain as the parent compound: for example, PGF₃.sub.α is 7-{3α,5α-dihydroxy-2β-[(3S)-3-hydroxy-trans-1,cis-5-octenyl]-1α-cyclopentyl}-cis-5-heptenoic acid.

Recent research has indicated that the prostaglandins are ubiquitous inanimal tissues and that prostaglandins, as well as analogues orderivatives thereof, have important biochemical and physiologicaleffects in mammalian endocrine, reproductive, central and peripheralnervous, sensory, gastro-intestinal, hematic, respiratory,cardiovascular, and renal systems.

In mammalian endocrine systems, experimental evidence indicatesprostaglandins are involved in the control of hormone synthesis andrelease in hormone-secreting glands. In rats, for example, PGE₁ and PGE₂increases release of growth hormone while PGA₁ increases growth hormonesynthesis. In sheep, PGE₁ and PGF₁.sub.α inhibit ovarian progesteronesecretion. In a variety of mammals, PGF₁.sub.α and PGF₂.sub.α areimplicated as luteolytic factors. In mice, PGE₁, PGE₂, PGF₁.sub.α andPGE₁.sub.β increase thyroid activity. In hypophysectomized rats, PGE₁,PGE₂ and PGF₁.sub.α stimulate steroidogenesis in the adrenal glands.

In the mammalian male reproductive system, PGE₁ contracts the smoothmuscle of the vas deferens. In the female reproductive system, PGE andPGF.sub.α compounds contract uterine smooth muscle. In general, PGE's,PGB's and PGA's relax in vitro human uterine muscle strips, whilePGF.sub.α 's contract such isolated preparations. PGE compounds ingeneral promote fertility in the female reproductive system whilePGF₂.sub.α has antifertility effects. PGF₂.sub.α also is believed to beinvolved in the mechanism of menstruation. In general, PGE₂ exertspotent oxytocic effects in inducing labor, while PGF₂.sub.α inducesspontaneous abortions in early pregnancy.

PGF.sub.α's and PGE's have been isolated from a variety of nervoustissue and they have been postulated to serve a neurotransmitter role.PGE₁ retards whereas PGF₂.sub.α facilitates transmission in motorpathways in the CNS. It has been reported that PGE₁ and PGE₂ inhibittransmitter release from adrenergic nerve endings in the guinea pig.

Prostaglandins stimulate contraction of gastrointestinal smooth musclein vivo and in vitro. In dogs, PGA₁, PGE₁ and PGE₂ inhibit gastricsecretion. PGA₁ exhibits similar activity in man.

In most mammalian respiratory tracts, PGE's and PGF.sub.α's relax invitro preparation of tracheal smooth muscle. In in vitro preparations,PGE₁ and PGE₂ relax human smooth muscle while PGF₂.sub.α contracts suchpreparations. PGE and PGF compounds are normally found in the humanlung, and it has been postulated that some cases of bronchial asthmainvolve an imbalance in the production or metabolism of those compounds.

Prostaglandins have been shown to be involved in certain hematicmechanisms in mammals. PGE₁, for example, inhibits thrombogenesis invitro through its effects on blood platelets.

In a variety of mammalian cardiovascular systems, PGE's and PGA's arevasodilators whereas PGF.sub.α's are vasoconstrictors, by virtue oftheir action on vascular smooth muscle.

Prostaglandins are naturally found in the kidney and reverseexperimental and clinical renoprival hypertension.

The clinical implications of prostaglandins and derivatives or analoguesthereof are far-ranging and include, but are not limited to thefollowing: in obstetrics and gynecology, they may be useful in fertilitycontrol, treatment of menstrual disorders, induction of labor, andhormone disorders; in gastroenterology, they may be useful in thetreatment of peptic ulcers, and various disorders involving motility,secretion, and absorption in the gastrointestinal tract; in therespiratory area, they may be beneficial in the therapy of bronchialasthma and other diseases involving bronchoconstriction; in hematology,they may have utility as anti-clotting agents in diseases such as venousthrombosis, thrombotic coronary occlusion and other diseases involvingthrombi; in circulatory diseases they may have therapeutic utility inhypertension, peripheral vasopathies, and cardiac disorders.

In general, the natural prostaglandins affect smooth muscle regardlessof origin in mammalian systems both in vivo and in vitro. This activityallows a rapid bioassay of prostaglandin derivatives or analogues by useof isolated muscle strips in vitro. (Cf. Bergstrom et al., Pharmacol.Rev., 20: 1 [1968]; Ferreira and Vane, Nature, 216: 868 [1961]).

The field to which this invention pertains is discussed in the followingreferences: The Prostaglandins, Val. I., P. Ramwell, Ed., New York,Plenum Press, 1973; Ann. N.Y. Acad. Sci., 180: 1-568 (1971); and Higginsand Braunwald, J. Am. Med. Assn., 53: 92-112 (1972).

SUMMARY

Prostaglandin analogues having the following structural formula IIIconstitute the subject matter of this invention: ##STR6## In formulaIII: J is hydrogen or hydroxyl; K is carbonyl or carbinol; L is ethyleneof vinylene; Q is hydrogen, loweralkyl of 1 to 3 carbon atoms, or apharmacologically acceptable nontoxic cation; and n is an integer havinga value of from 2 to 6 such that there exists between C₁₅ and C_(n) ₊ 16an alkylene bridge of n methylene groups. In III: carbon atoms arenumbered sequentially as indicated, following the conventional practiceutilized in prostaglandin chemistry; a dotted line ( ) represents acovalent bond projecting below the plane of a reference carbon atom(alpha-configuration) while a wedged line ( ) indicates a covalent bondprotruding above said reference plane (beta-configuration); and a swungdash or serpentine line ( ) denotes a covalent bond which can be ineither the alpha- or beta-configuration. In III, when the covalent bondat C₇ -C₈ is in the alpha-configuration and the bond C₁₂ -C₁₃ is in thebeta-configuration, the prefix "nat-" is used before the compound nameto describe this trans-orientation of the side-chains; however, thereverse case, i.e. when C₇ -C₈ is in the beta- and C₁₂ -C₁₃ is in thealpha-configuration, the prefix "ent-" is used before the compound name.Analogues or derivatives of PGE₁ and PGE₂ are represented by formula IIIin the case where J is hydroxyl, K is carbonyl, and L respectively isethylene or vinylene; similarly, analogues or derivatives of PGF₁ andPGF₂ are represented in the case where J and K are hydroxyl and Lrespectively is ethylene or ethenylene. Corresponding 11-desoxy-PGE and-PGF analogues or derivatives are indicated by both formulas when J ishydrogen.

An important structural feature of III is the presence of theunsaturated bond at C₁₃ and the hydroxyl substituent at C₁₅ both ofwhich are common to all natural prostaglandins. Unlike the latter,however, analogues with structure III contain the C₁₃ and the C₁₅ atomsin a cyclo alkylidene structure wherein the C₁₅ atom is incorporatedinto an n+ 3 carbon atom ring. In structure III, C₁₅ is the second atomin the ring.

The following are illustrative examples of formula III: ##STR7##

Compounds having formula III are prepared according to the synthesisreported by Sih et al. (J. Am. Chem. Soc., 95: 1676 [1973]), outlined inTable A. In the reaction sequences depicted in Table A, t-butyl-lithium(V), is commercially available or easily prepared according to methodswhich are well known in organic chemistry. Hexamethylphosphoroustriamide copper iodide, VI, is prepared as follows: (1) add 18.39 g ofpurified CuI (Inorg. Synth., 7: 9 [1963]) to 177 g KI in 135 ml. H₂ Oand stir with activated charcoal (Norite); (2) filter the solutionthrough infusorial earth (Celite) and add 14.5 g (0.089 mole) ofhexamethylphosphorous triamide (commercially available) under argonatmosphere; (3) filter, wash with aqueous KI and H₂ O; (4) dissolveproduct in dry ether, filter, remove ether in vacuo to obtain 13.85 ghexamethyl-phosphorous triamide copper (I) iodide NMR (COCl₃): singlet,δ 2.65. Compound VII can be prepared from2-(6-carbomethoxyhexyl)cyclopentane-1,3,4-trione (Cf. Katsube andMatsui; Agr. Biol. Chem., 33: 1078 [1969] for the synthesis of thiscompound) as described in the referenced Sih et al. publication.Examples of VIII which are employed in the synthesis of III and IVinclude:

2-(6-carbomethoxyhexyl)-4-hydroxy-2-cyclopenten-1-one;

2-(6'-carbomethoxy-cis-2'-hexenyl)-4-hydroxy-2-cyclopenten-1-one;

2-(6'-carbomethoxy-hexyl)-2-cyclopenten-1-one; and

2-(6'-carbomethoxy-cis-2'-hexenyl)-2-cyclopenten-1-one.

Compounds IV of Table A are synthesized according to the processreported by W. R. Benson and A. E. Pohland (J. Org. Chem., 29: 385[1964]):

                  TABLE A                                                         ______________________________________                                                ##STR8##                  (IV)                                         ##STR9##                                                                     (1) (CH.sub.3).sub.3CLi                                                              (V)                                                                           (2) [(CH.sub.3).sub.2 N].sub.3PCuI                                                                      (VI)                                                 ##STR10##                (VII)                                                ##STR11##                 (VIII)                                       ##STR12##                                                                           (4) CH.sub.3 CO.sub.2 H; THF; H.sub.2 O                                       (5) H.sub.2 O; THF; NaOH                                                       ##STR13##                  (III)                                      ______________________________________                                         ##STR14##     As defined in formula III, n has a value of from 2 to 6. Examples of     intermediates IV utilized in the reaction IV→→ III include     the following:

1-ethoxyethoxy-2-(2'-iodo-methylidene)-cyclopentane;

1-ethoxyethoxy-2-(2'-iodo-methylidene)-cyclohexane;

1-ethoxyethoxy-2-(2'-iodo-methylidene)-cycloheptane; and

1-ethoxyethoxy-2-(2'-iodo-methylidene)-octane.

Steps 4 and 5 in Table A involve removal of the ethoxyethoxy group andmethyl ester group, respectively.

In compounds having formula III, K can be converted from a carbonylgroup to a carbinol group by reaction with NaHB₄ in methanol.

Sodium or potassium salts of III can be prepared by known procedures.

The compounds represented by III are useful analogues of natural E- andF- prostaglandins and stimulate in vitro smooth muscle preparationsderived from a variety of tissues and organs of experimental animals.Such in vitro smooth muscle assays are widely utilized to determine theactivity of natural prostaglandins as well as prostaglandin analogues(Bundy et al., Ann. N.Y. Acad. Sci., 180: 76 [1961]; Bergstrom et al.,Pharmacol. Revs., 20: 1 [1968]). Details of the activity of certaincompounds having formula III are presented in Example 2, below.

Compounds III inhibit aggregation of human platelets in vivo and exhibituseful cardiovascular properties, as demonstrated in Example 3, below.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 A.2-(6'-Carboxyhexyl)-3-(2"-Anti-Hydroxymethylidenecycloheptyl)-Cyclopentanone

2.14 g (6.69 mmol) of 1-ethoxyethoxy-2-(2'-iodoethylidene)-cycloheptanewas dissolved in 40 ml anhydrous ether, cooled to -78° C., and stirredunder argon atmosphere. 11.0 ml of 1.23 M t-butyllithium in pentane wasadded and the mixture was stirred for 3 hours at -78° C. The reactionmixture was then added to a complex formed by dissolving 0.873 mg copper(I) pentyne in 23 ml ether with 2.43 ml of hexamethylphosphoroustriamide. The mixture was stirred at -78° C. resulting in an orangesolution. 1.34 g (6.0 mmol) of2-(6'-carbomethoxyhexyl)-2-cyclopenten-1-one in 23 ml of ether wasadded, the mixture was stirred for 15 min. at -78° C., and brought to 0°C. by means of an ice-salt bath over an interval of 1.5 hr. The mixturewas stirred for 0.5 hr. at 0° C. and an additional 0.5 hr. at 25° C. Themixture was processed with 20% aqueous (NH₄)₂ SO₄, 2% (V/V) 2H₂ SO₄ /H₂O, saturated aqueous NaHCO₃, saturated aqueous NaCl, filtered andstripped in vacuo to yield 2.10 g of a green oil. NMR (CDCl₃) analysisafforded the following data: δ 3.65, singlet, CO₂ CH₃ ; δ 4.70,multiplet, ##STR15## δ 5.32, doublet, ##STR16## J₁₂ ₋₁₃ = 9 Hz. Theexthoxyethoxy group was removed by reacting the product with 48 ml of65/35 acetic acid/water and 4.8 ml of tetrahydrofuran (THF) for 15 hrs.at 25° C. The product was processed with 20% (NH₄)₂ SO₄, 2% (V/V) H₂SO₄, saturated aqueous NaHCO₃, saturated aqueous NaCl, filtered andstripped in vacuo to yield 1.81 g of a yellow oil. NMR (CDCl₃) showed noabsorption at δ 4.70. The methyl ester was hydrolyzed by reaction with20 ml THF and 20 ml 1N NaOH for 18 hrs. at 25° C. The product wasprocessed as described above to obtain 1.44 g of a clear orange oil,2-(6'-carboxyhexyl)-3-(2"-anti-hydroxymethylidene-cycloheptyl)-cyclopentanone.

Analysis: IR: λ_(max) ^(CHCl).sbsp.3 2.78μ, 5.75μ, 5.85μ; MS: 336.318;NMR(COCl₃): δ 5.40, doublet, 1H, C₁₃ -H; J₁₂,13 = 9.0 Hz; δ 4.35,multiplet, 1H, CHOH; δ 7.86, broad singlet, 2H, CO₂ H, --OH;

B. Substitution of 1-ethoxyethoxy-2-(2'-iodo-ethylidene-cyclopropane,-cyclobutane, -cyclopentane, -cyclohexane, cyclooctane, or cyclononanein the above procedure yields the following 11-desoxy-PGE₁ analogues:

1.2-(6'-carboxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclopentyl)-cyclopentanone;

2.2-(6'-carboxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclohexyl)-cyclopentanone;

3.2-(6'-carboxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclooctyl)-cyclopentanone;and,

4.2-(6'-carboxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclononyl)-cyclopentanone.

C. Substitution of 2-(6'-carbomethoxy-cis-2'-hexene)-2-cyclopenten-1-onefor the corresponding 2-(6'-carbomethoxy-hexane)-2-cyclopenten-1-one inprocedures A and B results in the following 11-desoxy-PGE₂ analogues:

1.2-(6'-carboxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cyclopentyl)-cyclopentanone;

2.2-(6'-carboxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cyclohexyl)-cyclopentanone;

3.2-(6'-carboxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cycloheptyl)-cyclopentanone;

4.2-(6'-carboxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cyclooctyl)-cyclopentanone;and,

5.2(6'-carboxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cyclononyl)-cyclopentanone.

D. Substitution of2-(6'-carbomethoxyhexane)-4-hydroxy-2-cyclopenten-1-one or2-(6'-carboxymethyl-cis-2'-hexene)-4-hydroxy-2-cyclopenten-1-one inprocedures A and B yields the following PGE₁ and PGE₂ derivatives,respectively:

1.2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclopentyl)-4-hydroxy-2-cyclopenten-1-one;

2.2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclohexyl)-4-hydroxy-2-cyclopenten-1-one;

3.2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cycloheptyl)-4-hydroxy-2-cyclopenten-1-one;

4.2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclooctyl)-4-hydroxy-2-cyclopenten-1-one;

5.2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclononyl)-4-hydroxy-2-cyclopenten-1-one;

6.2-(6'-carbomethoxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cyclopentyl)-4-hydroxy-2-cyclopenten-1-one;

7.2-(6'-carbomethoxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cyclohexyl)-4-hydroxy-2-cyclopenten-1-one;

8.2-(6'-carbomethoxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cycloheptyl)-4-hydroxy-2-cyclopenten-1-one;

9.2-(6'-carbomethoxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cyclooctyl)-4-hydroxy-2-cyclopenten-1-one;and

10.2-(6'-carbomethoxy-cis-2'-hexene)-3-(2"-anti-hydroxy-methylidene-cyclononyl)-4-hydroxy-2-cyclopenten-1-one.

E. Hydride reduction of the PGE₁, PGE₂, 11-desoxy-PGE₁, and11-desoxy-PGE₂ analogues described in Sections B, C, and D, above, yieldthe corresponding PGF₁, PGF₂, 11-desoxy-PGF₁ and 11-desoxy-PGF₂analogues, respectively:

1.1-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclopentyl)-cyclopentane;

2.1-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclohexyl)-cyclopentane;

3.1-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cycloheptyl)-cyclopentane;

4.1-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cyclooctyl)-cyclopentane;

5.1-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclononyl)-cyclopentane;

6.1-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cyclopentyl)-cyclopentant;

7.1-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cyclohexyl)-cyclopentane;

8.1-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cycloheptyl)-cyclopentane;

9.1-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cyclooctyl)-cyclopentane;

10.1-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cyclononyl)-cyclopentane;

11.1,4-di-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclopentylidene-methyl)-cyclopentane;

12.1,4-di-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclohexylidene-methyl)-cyclopentane;

13.1,4-di-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cycloheptylidene-methyl)-cyclopentane;

14.1,4-di-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclooctylidene-methyl)-cyclopentane;

15.1,4-di-hydroxy-2-(6'-carbomethoxyhexane)-3-(2"-anti-hydroxy-methylidene-cyclononylidene-methyl)-cyclopentane;

16. 1,4-di-hydroxy-2-(6'-carbomethoxyhexene-3-(2"-anti-hydroxy-methylidene-cyclopentylidene-methyl)-cyclopentane;

17.1,4-di-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cyclohexylidene-methyl)-cyclopentane;

18.1,4-di-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cycloheptylidene-methyl)-cyclopentane;

19.1,4-di-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cyclooctylidene-methyl)-cyclopentane;and,

20.1,4-di-hydroxy-2-(6'-carbomethoxyhexene)-3-(2"-anti-hydroxy-methylidene-cyclononylidene-methyl)-cyclopentane.

EXAMPLE 2 Prostaglandin Activity in the Cascade Assay System

The superfusion technique introduced by Gaddum (Brit. J. Pharmacol., 6:321 [1953]) consists of dropwise bathing an isolated tissue with anutrient solution, instead of submersing it in a chamber filled with thefluid. This procedure allows a greater sensitivity for biologicalassays, since test compounds are less diluted than in usual systems. Anadditional advantage is that a compound can be tested simultaneously inseveral structures by arranging the tissues in vertical succession toallow successive contact with the test material. This procedure has beencalled the cascade system and has been specially useful fordetermination of prostaglandin activity (Ferreira and Vane, Nature, 216,868 [1967]); (Bergstrom et al., Pharmacol. Rev., 20: 1 [1968]).

A. Preparation of Tissues

Rat stomach fundus

After sacrifice of the animal, the stomach was removed, the antrum cuttransversely and the fundus cut in order to preserve the longitudinalmuscle as described by Vane (Brit. J. Pharmacol., 12: 344 [1959]).

Superfusion fluid

Krebs bicarbonte solution bubbled with a mixture of 95% O₂ and 5% CO₂ ata temperature of 37° C. was applied dropwise over the preparations at arate of 10 ml/min. The following antagonists were added to the solution:atropine (0.1 mcg/ml), phenoxy-benzamine (0.1 mcg/ml), propranolol (3.0mcg/ml), methysergide (0.2 mcg/ml) and brompheniramine (0.1 mcg/ml). Theuse of these antagonists in the nutrient eliminated the possibility ofsmooth muscle responses due to stimulation of cholinergic, adrenergic,serotonin or histamine receptors.

Test drugs

Prostaglandin derivatives were diluted in order to administerconcentrations ranging from 0.001 ng to 100 mcg. Concentrations wereapplied dropwise in a 0.5 ml-volume.

B. Results

Results of the cascade assay in the rat stomach fundus are shown inTable B for the following compound:2-(6'-carboxyhexyl)-3-(2"-anti-hydroxycycloheptylidenemethyl)-cyclopentanone.In Table B, a zero indicates no activity at the concentration tested.For comparison purposes, PGE₁ at a concentration of 31 ng produced 4.0 gof tension in the rat stomach fundus preparation.

                  TABLE B                                                         ______________________________________                                        TENSION        DOSE                                                           (grams)        (micrograms)                                                   ______________________________________                                        0              0.01                                                           0.6            0.10                                                           1.0            1.0                                                            2.1            10.0                                                           3.3            100.0                                                          ______________________________________                                    

EXAMPLE 3 A. Evaluation of Inhibition of Human Platelet Aggregation byAnalogues of Prostaglandins Human Structure III

The ability of test compounds to inhibit platelet aggregation wasdetermined by a modification of the turbidometric technique of Born(Nature, 194: 927 ([1962). Blood was collected from human volunteers whohad not ingested aspirin or aspirin-containing products within thepreceding two weeks in heparinized containers and was allowed to settlefor 1 hour. The platelet rich plasma (PRP) supernates were collected andpooled. Siliconized glassware was used throughout.

In a representative assay 1.9 ml of PRP and 0.2 ml of test compound atthe appropriate concentration (0.001 to 100 mcgm), or 0.2 ml ofdistilled water (control procedure) were placed in sample cuvettes. Thecuvettes were placed in a 37° C incubation block for 15 minutes, andthen in a spectrophotometer linked to a strip chart recorder. After30-60 seconds, 0.2 ml of a solution, prepared by diluting a calf-skincollagen solution 1:9 with Tyrodes' Solution, was added to eachcuvettes. Platelet aggregation was evidenced by a decrease in opticaldensity.

Calculation of the degree of inhibition of platelet aggregationexhibited by each concentration of test compound was accomplishedaccording to the method of Caprino et al., (Arzneim-Forsch., 23: 1277[1973]). An ED₅₀ value was then determined graphically,2-(6'-Carboxyhexyl)-3-(2"-anti-hydroxy-cycloheptylidenemethyl)cyclopentanonehas an ED₅₀ of 18.0 mcg/kg.

B. Evaluation of the Effects of Prostaglandin Analogues III on FemoralBlood Flow in the Dog

The peripheral vasodilator or constrictor effects of test compounds weredetermined in mongrel dogs of either sex, weighing between 10 and 20 kganesthestized intraveneously with 35 mg/kg of sodium pentobarbital. Anexternal iliac artery was dissected immediately above the femoral archfor a length of approximately 5 cm and a previously calibrated,non-connulating electromagnetic flowmeter sensor with a lumen between2.5 and 3.5 mm was placed snugly around the vessel. Cannulas were placedin a branch of the artery arising distally to the location of theflowmeter sensor for intraarterial drug administrations, in thecontralateral femoral artery for systemic blood pressures recordings andin the tracea for artificial respiration with room air. Femoral bloodflow and systemic blood pressure were continuously recorded with anelectromagnetic flowmeter and pressure transducer, respectively.

After an adequate control period, test compounds were injectedintraareterially at one log-spaced doses ranging from 0.001 to 10 mcg.,in a volume of 0.5 ml and at 5 to 10 minute intervals. Maximum changesin bloodflow, as well as any variations in blood pressure, weretabulated for each dose in absolute values (ml/min. and mmHg). Thecalculations were made taking as control values those existingimmediately before administration of each dose. The direction of theobserved change (plus for increase and minus for decrease) was alsonoted. The dose changing bloodflow by 100 ml/min (ED₁₀₀ ml/min.) wascalculated graphically.2'-(6'-Carboxyhexyl)-3-(2"-anti-hydroxy-cycloheptylidenemethyl)-cyclopentanonehas an ED₅₀ of 0.63 mcg/kg.

What is claimed is: 1.2-(6'-Carboxyhexyl)-3-(2"-anti-hydroxycycloheptylidenemethyl)-cyclopentanone.